First, abbreviations used in the specification are explained.
BPB: bromophenol blue
CMP: cytidine 5′-monophosphate
EDTA: ethylenediamine tetraacetic acid
EGTA: ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
Endo H: endo-β-N-acetylglucosaminidase H
ER: endoplasmic reticulum
FBS: fetal bovine serum
HRP: horseradish peroxidase
HPTLC: high performance thin-layer chromatography
LacCer: lactosylceramide
N-linked sugar chain: asparagine-linked sugar chain
N-linked glycoprotein: asparagine-linked glycoprotein
PBS: phosphate-buffered saline
PCR: polymerase chain reaction
PMSF: phenylmethanesulfonyl fluoride
PNGase F: peptide-N(4)-(N-acetyl-β-D-glucosaminyl) asparagine amidase
PVDF: polyvinylidene difluoride
SAT-I: sialyltransferase-I
hSAT-I: human SAT-I
mSAT-I: mouse SAT-I
zSAT-I: zebrafish SAT-I
SDS: sodium dodecyl sulfate
SDS-PAGE: sodium dodecyl sulfate-polyacrylamide electrophoresis
Further, in the specification, an amino acid X (one-letter notation) which is at the J-th position from the N-terminal of a protein is represented as “XJ”. For example, Asn at the 180th position from the N-terminal is represented as “N180” and Thr at the 336th position from the N-terminal is represented as “T336”.
Further, in the specification, a protein obtained by substituting an amino acid X (one-letter notation) at the J-th position from the N-terminal of a protein by an amino acid Z (one-letter notation) is represented as “XJZ”. For example, a protein in which His at the 177th position from the N-terminal is substituted by Asp is represented as “H 177D” and a protein in which Asn at the 224th position from the N-terminal is substituted by Lys is represented as “N224K”. Further, when substitution of one amino acid by another occurs at a plurality of sites, the substitutions are represented side by side. For example, a protein in which His at the 177th position from the N-terminal is substituted by Asp and Asn at the 224th position from the N-terminal is substituted by Lys is represented as “H177D, N224K”.
Most of secretory proteins in a living body are considered to be present as glycoproteins. Sugar chains have a wide variety of functions such as physical stabilization of proteins, expression of enzyme activity, cell adhesion, metastasis of cancer, signal transduction, subcellular localization, microbial infection, and immune response. Therefore, lack of the sugar chains to be linked to glycoproteins, in many cases, causes effects such as failure or reduction of expression of physiological activity of the glycoprotein.
An N-linked sugar chain, which is a sugar chain to be linked to a glycoprotein, is known to link to Asn in a consensus sequence comprising Asn-Xaa-Ser/Thr (provided that Xaa is an amino acid other than Pro) of a protein.
It is also known that a glycosyltransferase is one of glycoproteins, and a plurality of N-linked sugar chains are linked to most of glycosyltransferases. It has been reported that a glycosyltransferase wherein N in said consensus sequence is substituted by Q, which is structurally most similar to N, and a glycosyltransferase treated with tunicamycin, which inhibits formation of dolichol pyrophosphate-N-acetylglucosamine to suppress sugar transfer to a protein, have a remarkably reduced enzymatic activity (Non-patent Document 1 and Non-patent Document 2).
For example, α1,3-fucosyltransferase (Fuc) III, IV, V and VI, α2,3-sialyltransferase (ST3Gal I), α2,6-sialyltransferase (ST6Gal I), α2,8-sialyltransferase (ST8Sia I); GD3 synthase, UDP-N-acetylglucosamine:β-D-mannoside β1,4-N-acetylglucosaminyltransferase III (GnT III), core 2β1,6-N-acetylglucosaminyltransferase (C2 GnT), galactosylceramide sulfotransferase (CST), N-acetylgalactosaminyltransferase 1 (GalNac-T), β1,3-galactosyltransferase (Gal-T2), UDP-glucuronosyltransferase 2B (UGT2B) and the like are reported to require sugar chains to express their enzymatic activities.
SAT-I is one of sialyltransferases (i.e., sialic acid transferases) that transfer sialic acid to lactosylceramide and synthesizes GM3, and hSAT-I, mSAT-I and zSAT-I have been cloned (Non-patent Document 3 and Non-patent Document 4). In addition, SAT-Is from dog, bovine, rat, chicken, medaka, and tetradon have been cloned.
However, in N-linked glycoproteins, a mutant protein is not known which has no N-linked sugar chains to be linked thereto, but retains a physiological activity of a glycoprotein before the mutation is introduced. Neither is a method of producing such a mutant protein and the like.    Non-patent Document 1: Martina J. A. et al., 1998, The Journal of Biological Chemistry, vol. 273, p. 3725-3731    Non-patent Document 2: Eckhardt M. et al., 2002, The Biochemical Journal vol. 368, p. 317-324    Non-patent Document 3: Ishii A. et al., 1998, The Journal of Biological Chemistry, vol. 273, p. 31652-31655    Non-patent Document 4: Kono M. et al., 1998, Biochemical and Biophysical Research Communications, vol. 253, p. 170-175